The experiments described in this proposal are designed to provide insights into the mechanism and regulation of pre-mRNA polyadenylation. The following five specific aims are proposed. 1. Purification and characterization of polyadenylation factors. Efforts to purified CF1 and especially CFII from HeLa nuclear extract will be continued. cDNA clones will be isolated and recombinant proteins and antibodies produced. Studies to understand the role of hPTS will be continued, and the possibility that additional polypeptides are required for 3' cleavage will be explored. 2. Analysis of protein-protein and protein- RNA interactions. Studies to understand the RNA binding specificities of CstF-64 and its yeast homologue RNA15 will be continued. The region of CPSF-160 responsible for AAUAAA binding, which appears to represent a novel RN binding motif, will be identified. Protein-protein interactions will be investigated, concentrating on the subunits of CstF, which all contain interesting domains. The possible existence and significance of overlapping for identical binding sites in CstF-64 for CstF-77 and hPTA will be investigated. In vitro protein binding assays will be employed when the interacting proteins 3. In vivo analysis of polyadenylation. Studies employing the chicken B cell line DT40 will be continued. A cell line with both alleles of CstF-64 disrupted, containing a tetracycline-repressible cDNA as the only source of CstF-64, will be further analyzed. The basis and specificity of a dramatic decrease in IgM H-chain mRNA accumulation will be determined and the effects of varies CstF-64 concentrations on alternative ploy (A) site selection studied further. Efforts to construct a similar line containing tet-repressible PAP will continue. This line will be used to investigate the functional significance of PAP isoforms produced by alternative splicing, and the requirement of PAP hyperphosphorylation during the cell cycle. 4. Regulation of polyadenylation. Studies examining the possible ability of CPEB to enhance polyadenylation by stabilizing CPSF binding through a direct interaction with CPSF-160 will be continued. Experiments to understand the basis for regulation of PAP activity by p34 cdc2/cyclin B phosphorylation will be continued, including analysis of the role of direct cyclin binding to PAP and the mechanism by which hyperphosphorylation inhibits PAP activity. The effects of PAP phosphorylation on protein binding will be explored, using the well-define U1A-PAP interaction as a model. 5. Polyadenylation and transcription. Recent studies suggesting a possible link between transcription and polyadenylation will be continued, first by establishing a coupled transcription-polyadenylation system. The importance of CPSF recruitment by TFIID for efficient 3' end formation will be investigated, as will the possibility that dissociation of CPSF from RNA polymerase II as the poly (A) signal contributes to subsequent transcription termination. Finally, the CstF-64 tet-repressible cell line will be used to determine if depletion of CstF-64 affects splicing of a poly(A) site proximity intron, transcription termination downstream of the poly (A) site, and/or transcription initiation.